Lipid extract having anti-inflammatory activity

ABSTRACT

Anti-inflammatory, and particularly anti-arthritic, treatment of a human or animal patient comprises administration to the patient of an effective amount of a lipid extract of  Perna canaliculus  or  Mytilus edulis.

This is a continuation of U.S. application Ser. No. 09/029,957, filedMay 5, 1998, now U.S. Pat. No. 6,083,536, a national phase filing basedon PCT/AU96/00564, filed Sep. 10, 1996, priority benefit based on saidearlier filings as well as Australian Appln. No. 5311/95, filed Sep. 11,1995 being claimed herein.

FIELD OF THE INVENTION

This invention relates in general to a preparation havinganti-inflammatory, and particularly anti-arthritic, activity which is alipid extract of mussels, including the New Zealand green lipped mussel,Perna canaliculus, and the blue mussel Mytilus edulis.

BACKGROUND OF THE INVENTION

There is at the present time a significant medical need for newanti-inflammatory and anti-arthritic drugs with reduced side effects andprolonged in vivo activity and in particular for compounds which willmoderate the progress of the arthropathies. Plants and other livingcells offer a vast reservoir of compounds which have pharmacologicaleffects on humans. Natural products have frequently been the source ofeffective drugs and lately there has been an increased interest in theanalysis of these natural products, especially where a clinical benefitis claimed. Marine organisms contain metabolites that can act aspharmacological agents and aid in the treatment of inflammation.

An anti-inflammatory activity of Perna canaliculus (New Zealand GreenLipped Mussel) was first implicated as part of a pharmacological studyon leukaemia. Initial assessment of the anti-inflammatory activity ofPerna canaliculus was first attempted using a polyarthritis model inrats¹. These studies however failed to show the presence of anysignificant anti-inflammatory activity in the mussel preparation. Incontrast, Miller and Ormrod² using a carrageenin-induced paw oedemaassay³, were able to show that mussel preparations, when administeredintraperitoneally, gave a significant reduction in the swelling of acarrageenin-induced rat paw oedema. Subsequently, they fractionated anon-dialysable, water-soluble fraction from the mussel preparation thatpossessed anti-inflammatory activity. The aqueous extract showed adose-ependent anti-inflammatory activity when administeredintraperitoneally and could not be detected upon oral administration ofthe mussel powder. It was suggested that the water-soluble fractiontherefore contained an irritant component possessing apparentanti-inflammatory activity.

Rainsford and Whitehouse⁴ also reported that freeze-dried powderedpreparations of the whole mussel given orally to rats showed some modestanti-inflammatory activity in the carrageenin-induced paw oedema assay,and that this material strikingly reduced the gastric ulcerogenicity ofseveral non-steroidal anti-inflammatory drugs in rats and pigs.

Use of the whole mussel extract in the treatment of both rheumatoidarthritis and osteoarthritis in human patients has also been reported⁵.

Initial work leading to the present invention based on lipid extractsfrom Perna canaliculus powder prepared using solvent extractiontechniques (in contrast to earlier work on aqueous fractions),established that the lipid fractions show a measure of anti-inflammatoryactivity when tested in appropriate model systems. A reliable source oflipid extract of Perna canaliculus and Mytilus edulis has subsequentlybecome available through the procedure of supercritical fluid extraction(SFE). The lipid extract is obtained as a dark yellow-brown viscous oilexhibiting strong ultraviolet absorbing character which is consistent inphysical data to lipid extracts obtained from earlier solvent extractionprocedures.

SUMMARY OF THE INVENTION

Accordingly to one aspect, the present invention provides a method ofanti-inflammatory treatment of a human or animal patient, whichcomprises administration to the patient of an effective amount of alipid extract of Perna canaliculus or Mytilus edulis.

In another aspect, the present invention provides an anti-inflammatorycomposition comprising a lipid extract of Perna canaliculus or Mytilusedulis as an active component thereof, together with one or morepharmaceutically acceptable carriers and/or diluents.

In yet another aspect, the invention extends to the use of a lipidextract of Perna canaliculus or Mytilus edulis in the preparation of acomposition for anti-inflammatory treatment of a human or animalpatient.

DETAILED DESCRIPTION OF THE INVENTION

The terms “anti-inflammatory treatment” and “anti-inflammatorycomposition” as used herein, relate to treatment of, or compositions fortreatment of inflammatory conditions in general, including arthriticconditions such as osteoarthritis and rheumatoid arthritis, as well asin treatment of multiple sclerosis and various viral infections.Activity of a compound for use in such treatment may be demonstratedusing standard assays, for example using a carrageenin-induced pawoedema assay or by the ability to beneficially limit the onset orprogression of an experimental polyarthritis, as described in detailherein.

Preferably, the lipid extract which is used in the treatment orcomposition of the present invention is an extract prepared bysupercritical fluid extraction (SFE) of freeze-dried powdered musselusing a cryogenic fluid (such as cryogenic fluid CO₂) as the extractingmedium. In comparison to solvent extraction techniques, supercriticalfluid extraction using cryogenic fluid CO₂ produces a lipid extract richin non-polar lipids, particularly in free fatty acids. While the exactcomposition of the lipid extract has not yet been established, it isknown to contain not only free fatty acids (including unsaturated fattyacids), but also triglycerides and cholesterol esters.

A variety of administration routines are available. The particular modeselected will depend, of course, upon the particular condition beingtreated and the dosage required for therapeutic efficacy. The methods ofthis invention, generally speaking, may be practised using any mode ofadministration that is medically acceptable, meaning any mode thatproduces therapeutic levels of the active component of the inventionwithout causing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, topical, nasal, transdermal orparenteral (e.g. subcutaneous, intramuscular and intravenous) routes. Inparticular, the lipid extract of the present invention has been found tobe active when administered orally, subcutaneously and transdermally.

Transdermal administration of the lipid extract is a particularlypreferred administration mode, as the lipid extract has been found tohave surprising anti-inflammatory activity when administeredtransdermally.

The compositions of this invention may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Such methods include the step of bringing the activecomponent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active component into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the activecomponent, in liposomes or as a suspension in an aqueous liquid ornon-liquid such as a syrup, an elixir, or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active component which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using thosesuitable dispersing or wetting agents and suspending agents. A sterileinjectable preparation may be formulated as a sterile injectablesolution or suspension in a nontoxic parenterally-acceptable diluent orsolvent, for example as a solution in polyethylene glycol and lacticacid. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed including synthetic mono-or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Compositions suitable for transdermal administration convenientlycomprise the active component in an ointment or lotion base or vehicle,and may include a skin penetration enhancing agent to assist inadministration of the active component. Suitable bases or vehicles areoils such as olive or emu oil, administered alone or with a penetrantsuch as cineole or limonene.

Other delivery systems can include sustained release delivery systems.Preferred sustained release delivery systems are those which can providefor release of the active component of the invention in sustainedrelease pellets or capsules. Many types of sustained release deliverysystems are available. These include, but are not limited to: (a)erosional systems in which the active component is contain within amatrix, and (b) diffusional systems in which the active componentpermeates at a controlled rate through a polymer.

The formulation of such therapeutic compositions is well known topersons skilled in this field. Suitable pharmaceutically acceptablecarriers and/or diluents include any and all conventional solvents,dispersion media, fillers, solid carriers, aqueous solutions, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and it isdescribed, by way of example in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activecomponent, use thereof in the pharmaceutical compositions of the presentinvention is contemplated. Supplementary active ingredients can also beincorporated into the compositions.

Oral or transdermal administration will be preferred for many conditionsbecause of the convenience to the patient, although localised sustaineddelivery may be more desirable for certain treatment regimens.

The active component is administered in therapeutically effectiveamounts. A therapeutically effective amount means that amount necessaryat least partly to attain the desired effect, or to delay the onset of,inhibit the progression of, or halt altogether, the onset or progressionof the particular condition being treated. Such amounts will depend, ofcourse, on the particular condition being treated, the severity of theconditions and individual patient parameters including age, physicalcondition, size, weight and concurrent treatment. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is preferred generally that amaximum dose be used, that is, the highest safe dose according to soundmedical judgement. It will be understood by those of ordinary skill inthe art, however, that a lower dose or tolerable dose may beadministered for medical reasons, psychological reasons or for virtuallyany other reasons.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the human or animal patients to be treated; eachunit containing a predetermined quantity of active component calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier and/or diluent. The specifications forthe novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activecomponent and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecomponent for the particular treatment.

Generally, daily doses of active component will be from about 0.01 mg/kgper day to 1000 mg/kg per day. Small doses (0.01-1 mg) may beadministered initially, followed by increasing doses up to about 1000mg/kg per day. In the event that the response in a subject isinsufficient at such doses, even higher doses (or effective higher dosesby a different, more localised delivery route) may be employed to theextent patient tolerance permits. Multiple doses per day arecontemplated to achieve appropriate systemic levels of the activecomponent.

Throughout this specification unless the context requires otherwise, theword “comprise”, or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

Further features of the present invention are more fully described inthe following Example(s). It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

EXAMPLE 1 A PREPARATION OF LIPID EXTRACT

A.1 Raw Material

The green lipped mussel (Perna canaliculus) is harvested on the southcoast of New Zealand at which time the total mussel is stabilised withtartaric acid. Freeze drying results in a dry power of pulverised form.

A.2 Extraction of Lipids

The technique of supercritical fluid extraction (SFE) is utilised toextract the biologically active lipids from the crude mussel powder.Cryogenic fluid CO₂ is used as the extracting medium. The CO₂ isexpanded to atmospheric pressure and the extract is presented as aconcentrated oil. The powder yields 3-3.5% of oil.

A.3 Profile of the crude oil

The extractable oil is orange amber in colour and is a viscous liquid atambient temperature. The extract is stored below 4° C. and is handled ina nitrogen atmosphere. The crude oil shows strong UV activity and isprotected from light to minimise the polymerisation of double bondcomponents.

PILOT SCALE SUPERCRITICAL FLUID EXTRACTION

Extraction of total lipids in freeze-dried mussel powder, Pernacanaliculus was performed on a pilot scale SFE unit undertaken at theFood Research Institute (Department of Agriculture, Werribee, Vic.,Australia).

B.1 Instrumentation

Extractions were performed on a pilot scale extraction unit consistingof five basic sub-units (Distillers MG Limited., England, UK). The fivebasic units comprise: Carbon dioxide supply, Solids extraction, Primaryseparation, Evaporation and Tailing units.

The carbon dioxide supply unit consists of two CO₂ cylinders connectedin parallel and placed on a weighing scale for recharging whenappropriate. The extraction unit can be supplied with liquid SC—CO₂ andSC—CO₂. For this work the SFE unit was operated using SC—CO₂. Solidmaterial was placed in the leaching column and the primary separatorfacilitates separation of extracted material by reduction of pressure(which allows extract to settle), adsorption or liquid extraction. Thefluid extract was passed into the evaporation unit to evaporate the CO₂by the use of internal heating tubes. The vapour may contain volatilesand thus it is subsequently passed to the tailing column to be scrubbedby pure liquid CO₂. The tailing unit traps the gaseous CO₂ from theevaporator unit and returns the volatile components to the evaporator.

B.2 Pilot plant extraction procedure

Mussel power (300 g) was charged to the extraction unit (leachingcolumn). SC—CO₂ was delivered at a flow rate of 3.0 kg/h for two hoursper extraction. Extractor temperature was set at 40° C. and theextractor pressure at 310 bar (4,500 psi). The evaporator temperaturewas held constant at 40° C. The mussel lipid extracts were stored undernitrogen at −10° C. In amber glass sealed containers.

EXAMPLE 2

Lipid extract of Perna canaliculus (herein referred to as “PCO”)prepared by SFE as described in Example 1 was assayed for acuteanti-inflammatory activity in the standard carrageenin rat oedema assay,as well as for anti-arthritic activity (measuring activity insuppressing chronic inflammation) against experimentally inducedpolyarthritis in rats⁶.

Comparative tests were carried out with Seatone™, a commerciallyavailable freeze dried powder preparation of Perna canaliculus.

A. METHODS

PCO was kept at −20° C. Traces of hexane etc. were not removed beforeuse. Formulations were aged no more than 24 hours at 4° C.

A.1 Anti-inflammatory Assay

Female Wistar rats (180-220 gm) were pre-dosed p.o. or i.p. with testformulations. 40 minutes later they were injected in each rear paw with0.1 ml saline containing 0.6 mg Na carrageenin. The subsequent pawoedema was quantified by measuring the increase in paw thickness afterone and two hours with a screw gauge micrometer. The ED₅₀ for aspirin isapproximately 150 mg/kg in this acute assay. [The oedema recedes afterthree hours with this dose of carrageenin.]

A.2 Anti-Arthritic Assay

Though more time consuming, this assay is of more value than the acuteassay (described above) as it detects not only (i) thoseanti-inflammatory drugs acting in the acute assay (but often at lowerED₅₀ values) but also (ii) other agents e.g. clobuzarit (CIoZic^(R)) orlobenzarit (CCA), which obviously have unique arthritis-suppressantproperties but are not anti-oedemic/antipyretic or otherwise detectablein acute assays.

The protocol used is to pre-establish disease by injecting thearthritigen on Day 0, look for first signs of paw inflammation (usuallymanifest on Days 10-12) and then dose the animals with test materialsfor four days. Over this period, the paw inflammation in the untreatedcontrol animals “blooms” rapidly to reach a near maximum on Day 14.Drugs acting over this time-frame to hold down the paw swelling arecertainly anti-symptomatic but rarely disease-ablating. On ceasingtreatment, there is usually a recrudescence in symptoms. This however isa positive feature of this assay as it clearly indicates a) the drug hasa finite action but gives some idea of its duration of efficacy by therapidity (or otherwise) of the rebound; and b) the animals with minimalsigns of disease on Day 14 reflected a positive drug effect rather thanfailure to respond to the original arthritigen (i.e. false negatives).An accurate description of this assay is “late prophylaxis”. Obviouslyby delaying treatment until after Day 14, one can look for “therapeuticactivity”, but since changes may be slow or negligible within four days(even with some powerful NSAIDs), collection of adequate data isdifficult.

Details of the protocol used are: inject the arthritigen into thetailbase of female Wistar rats (160-200 gm)=800 mcg heat-killed Mycobacttuberculosis suspended in 0.1 ml squalane in such a fashion as to avoidany blood vessels and to promote optimal drainage into the lymphatics.Ten days later, the animals were weighed, the thickness of the rear pawsand tail was measured and signs of inflammation in the forepaws scoredon a scale of 0 to 4 +. After dosing with test compounds once daily forfour days (i.e. on Days 10 through 13 post-arthritigen), thesemeasurements were repeated on Days 14 and 18 (i.e. after completion oftreatment and again after four days rebound) to monitor the arthritisdevelopment. For transdermal administration of test formulations,animals were shaved over the back of the neck under light anaesthesia(Forthane) on Day 1 0, to expose an area of skin=ca.6cm². Formulationswere applied once daily with rubbing for up to three minutes in avolume=2.5 ml/kg (i.e. approximately 0.5 ml/rat). The first applicationwas given six hours after shaving to be sure there was no skin abrasionsbefore treatment.

Antipyretic Assay

Young rats (less than 160 gm) were inoculated with 2 gm/kg driedbrewer's yeast suspended in saline at 11 pm. At 8 am, their rectaltemperatures were measured. Those showing a stable fever (temperaturegreater than 39.2° C.), at 9 am, were then dosed with test compounds.Paracetamol, 150 mg/kg was used for reference.

B. RESULTS

Acute Anti-inflammatory Activity (Table 1)

Table 1 indicates that neither Seatone nor PCO expressed anti-oedemicactivity in the short-term assay. Co-administration with a synergist hadno effect while amplifying that of Ibuprofen.

Anti-Arthritis Assay (Table 2)

Table 2 shows the results of the first two tests of PCO applied toarthritic rats either orally (p.o.) or subcutaneously (s.c.) or in atransdermal application (t.d.) at the one dose of 50 mg/kg given forfour successive days.

Diluting PCO into refined commercial olive oil (Vetta) allowed the samestock solution to be tested in all three modes of delivery(p.o./s.c./t.d.). The olive oil (OO) base largely excludes oxygen andcontains little metal catalyst and therefore has “keeping” propertiesfor many unstable (unsaturated) compounds.

The transdermal formulations were prepared from the OO stock by addingskin penetration enhancers (PE) in the relatively high proportion of 20%v/v. The PE's used were cineole (=eucalyptol), methyl salicylate (=oilof wintergreen) or isopropanol (=rubbing alcohol). The PCO-salicylategroup was perhaps the least responsive which might indicate some(negative) interaction between this salicylate ester and the activeprinciple(s) of PCO.

TABLE 1 Anti-Inflammatory activity in the acute Carrageenin paw oedema.n = 2 rats/group MPL = Misoprostal, 0.5 mg/kg p.o. as synergist. %Inhibition Oedema mg/kg Test Materials MPL 1 hr 2 hr 300 SEATONE ®, p.o.− 08 (−12)* + 15 20 50 PCO-Tween, p.o. − 07 04 + (−04)  02 —, i.p. −(−12)  06 + 04 15 30 IBUPROFEN − 37 42 + 56 58 *(neg. values) = >controls

Conclusions:

(i) Both PCO and Seatone are inactive, even with synergist.

(ii) Assay responds to a standard NSAID and this particular synergist.

TABLE 2 Anti-Inflammatory activity in arthritic rats. n = 4 rats/group.Test compounds administered in 4 successive daily doses. Mean increasein* Wt % Inhib Dose/kg Treatment Rear paw Tail Fore paw (gm) Rear FrontRebound** A - Oral (p.o.) or Subcutaneous (s.c.) application: — None0.90 mm 0.24 mm 1.1+ 05 — — 0 2.5 ml Olive oil, 0.87 0.30 + 03 03% 09% 0s.c. 50 mg PCO- 0.11 0.04 0.1+ 10 88 91 + Olive oil s.c. p.o. 0.06 −0.100.4+ 05 93 64 2+ 50 mg PCO- 0.03 −0.15 0.3+ 09 97 83 + Tween p.o. B =Transdermal application: — None 1.08 0.17 1.5+ 01 — — 0 2.5 ml Oliveoil-Cineole 0.96 0.05 1.4+ 08 11% 07% 0 50 mg PCO in 00- 0.12 −0.61 0 0489 100 2+ Cineole PCO in 00-Iso Pr −0.01 0.09 0.1+ −01 100 93 2+ OH PCOin 00-Me Sal 0.22 0.23 0.4+ 05 80 73 2+ *over days 10 → 14 **over days14 → 18 Vehicles = Olive oil, 4 vol:Penetrant, 1 vol.

Conclusions:

(i) PCO active at 50 mg/kg in 6 different modes of administration.

(ii) Vehicle controls (olive oil alone/with cineole) had insignificanteffect on arthritis.

EXAMPLE 3

This Example sets out the results of further experiments onanti-arthritic and acute anti-inflammatory activity of the lipid extractof Perna canaliculus (PCO), showing it to be active down to 10 mg/kg,and possible even at 2.5 mg/kg, given either orally or transdermally inan olive oil vehicle.

Anti-arthritic Activity

Two experiments were set up to evaluate PCO at lower doses than the 50mg/kg level found to show anti-inflammatory activity in arthritic ratswhen given p.o. or t.d. (see Example 2).

In the first experiment (Part A, Table 3) the olive oil formulationshowed activity down to 2.5 mg/kg given orally. There was a significantrebound over the following four days, on ceasing dosing, in all threetreatment groups.

In the second experiment (Part B, Table 3) using transdermaladministration, the olive oil formulation “thinned” with 20% cineole(v/v) showed good activity down to 10 mg/kg. The result at 2.5 mg/kg isonly provisional as it is not clear that there was a full “rebound”(possibly indicating some animals were “false-positives”=low reactors tothe original arthritogen).

Alternative dermal formulations based on olive oil with 20% D-limoneneor a non-oily vehicle (isopropanol with 20% v/v propylene glycol—thislast being added to minimise skin dehydration) were clearly lesssatisfactory than the original olive oil-with-cineole delivery system.The alcohol vehicle would probably have facilitated decomposition of PCOin contrast to the olive oil.

Anti-inflammatory Activity

Kaolin-induced paw oedema was investigated as a possible assay for PCOsince this inflammation is slower developing than that induced withcarrageenin (which recedes after three hours). The protocol involvesinjecting 5 mg kaolin, suspended in water (not saline, in which itaggregates), into each rear paw 40 minutes after dosing the rats withPCO given both p.o. or i.p. as dispersions prepared with 0.02% Tween ineither water (p.o.) or saline (i.p.). The paw swelling was then read twohours and five hours after the kaolin injection.

The results from one experiment (Table 4) with only two rats per groupwas ambiguous: the oral application may have provided some anti-oedemicactivity. The i.p. PCO/saline emulsion was physically less satisfactoryand seems to have been inactive.

TABLE 3 Further studies of anti-inflammatory action of PCO in arthriticrats. N = 4 rats/group PCO Mean Swelling Inhibition Treatment (mg/kg)Rear paw Tail Front paw Rear Front A. By oral administration. None 1.37mm 0.14 mm 1.5 + mm — — PCO Olive oil 25 −0.12 mm −0.24 mm 0.2 + mm100%  87% 10 0.04 mm −0.41 mm 0.5 + mm 97% 67% 2.5 0.40 mm +0.23 mm0.5 + mm 70% 67% B. By Dermal application. Olive Oil-Cineole* — 0.82 mm0.06 mm 0.8 + mm — — 25 −0.12 mm −0.27 mm 0 100%  100%  10 0.38 mm −0.01mm −0.1 + mm 54% 100%  — 2.5 0.39 mm −0.22 +0.1 + mm 52% 87% OliveOil-Limonene* 50 0.53 0.56 1.1 + mm 35%  0% iPA-PrG** 50 0.63 0.01 0.8 +mm 23%  0% *= 4:1 v/v, 2.5 ml/kg **= 4 vol. Isopropanol with 1 vol.Propylene glycol.

Conclusions:

(i) PCO active at 10 mg/kg p.o. or t.d.

(ii) PCO may be active ever at 2.5 mg/kg

(iii) Cineole>Limonene for t.d. use with PCO

(iv) Non-oil vehicle seems inappropriate for t.d. delivery of PCO.

TABLE 4 Kaolin-induced paw oedema in female Wistar rats. N = 2 rats/gponly SYN = Synergist, Misoprostol @ 1 mg/kg p.o. % Inhibition OedemaMg/kg Treatment Syn 2 HR 5 HR 150  Aspirin p.o. − 55 51 — None + −15 0750 PCO, p.o. + 22 41? 50 PCO, i.p. + −10 09

Conclusions:

(i) PCO, p.o. may show delayed activity in this anti-inflammatory assay.

(ii) PCO, i.p. did not.

EXAMPLE 4

This Example demonstrates the effect of the lipid extract of Pernacanaliculus (PCO) and various other oils available on the retail healthfood market when administered prophylactically to female Wistar ratsdeveloping adjuvant-induced polyarthritis (see Example 2). Treatment wascarried out in six groups (n=5 rats per group) as follows:

A: Untreated control group

B: Flax Oil (Barleaus); 2000 mg/kg body weight/day.

C: Evening Primrose Oil (Efamol); 2000 mg/kg body weight/day.

D: Norwegian Salmon Oil (J. R.Carlson); 2000 mg/kg body weight/day.

E: MAXEPA (Solgar); 2000 mg/kg body weight/day.

F. PCO; 20 mg/kg body weight/day.

The results are shown in Table 5 where the swelling of the arthriticpaws is expressed as a percentage of the untreated control group A.These results show that PCO is 200× more potent than EPA, and 350× morepotent than Evening Primrose Oil.

TABLE 5 Increased paw % Dose rate Group Treatment diameter inhibition(mg/KG) A Untreated control 1.09 mm 0% 0 group B Flax Oil 1.07 mm 2%2000 C Evening Primrose Oil 0.82 mm 25% 2000 D Norwegian Salmon Oil 0.74mm 32% 2000 E MAXEPA 0.55 mm 50% 2000 F PCO 0.23 mm 79% 20

EXAMPLE 5

This Example shows therapeutic treatment of arthritis inflammation inrats using PCO, unstabilised whole mussel extract (GL Mussel NZ),stabilised whole mussel extract (GL Mussel Aust), celery seed oil andindomethacin. Rats with the first signs of arthritis 10 days afterinoculating with mycobacterial arthritogen were then treated orally withthe test materials for 4 days only. Effectiveness of the test materialswas measured as increased rear paw thickness on day 14 when compared tothe control group H.

The results are shown in Table 6 and indicate that PCO is more effectivethan indomethacin in this model.

TABLE 6 % Reduction in Treatment Dose Rate mg/kg Inflammation A. PCO 591 B. GL Mussel (NZ) 300 21 C. GL Mussel (Aust) 300 97 D. Celery SeedOil 3000 12 E. Indomethacin ** 5 83 F. Indomethacin 3 68 G. Indomethacin1 26 H. No treatment 0 ** Toxic at this dose rate.

EXAMPLE 6

This Example provides further data comparing the effectiveness of thelipid extract of Perna canaliculus (PCO) when compared with freeze-driedwhole mussel powder (Seatone™) and ibuprofen (Nurofen™), tested againstcollagen (II)-induced auto-allergic arthritis in rats. A gastro-toxicityassay was also carried out on the test materials.

Anti-arthritic Assay.

Collagen (Type II) induced polyarthritis was induced in female Wistarrats by injection of collagen (II) with a non-arthritogenic adjuvant tosensitise the test animals.

Table 7 shows results obtained in therapeutic (i.e. delayed) treatmentwith the test materials. The treatment was delayed until day 8 at thefirst signs of arthritis, then continued till day 14 (i.e. 7 dailydoses) with the arthritis assessed on day 15 and again on day 18 —thelatter indicating rebound on ceasing dosing. PCO was given orally afterdilution into olive oil (8 mg/ml) and administered at 2.5 ml/kg/day togive 20 mg/kg.

The results show that PCO at 20 mg/kg is as effective in therapeutictreatment as 300 mg/kg stabilised mussel extract (Seatone) and 50 mg/kgibuprofen (Nurofen).

Table 8 shows results obtained in both prophylactic and therapeutictreatment with the test materials. Prophylactic treatment was from day 1to day 13 (i.e. total 15 doses) orally (p.o.). Therapeutic treatment wasfrom day 9 to day 13 (i.e. total 5 doses) transdermally (t.d.). PCO wasdiluted into olive oil (6 mg/ml) for p.o. dosing at 2.5 ml/kg/day togive 15 mg/kg, and diluted into olive oil-cineole (17.3 v/v) (10 mg/ml)for t.d. administration at 2 ml/kg/day to give 20 mg/kg.

This experiment also included sodium aurothiomalate (ATM=Mycocrysin®)given as a reference anti-arthritic drug, administered every second day(total=8 doses) subcutaneously in saline at 6.3 mg/kg (higher doses weretoxic). The relatively high weight gain of animals treated with ATM maybe misleading, perhaps being a sign of incipient kidney damage (impairedurination), rather than a beneficial reduction in the normal weight lossassociated with chronic inflammation.

Seatone™ was administered p.o. at 300 mg/kg; Nurofen™ was administeredp.o. at 50 mg/kg; Na Aurothiomalate (ATM) was administered in 8 doses at6.3 mg/kg subcutaneously (s.c.).

The results show that PCO is particularly effective when administered

Gastro-toxicity Assay

Gastro toxicity studies were carried out on female Wistar rats withestablished arthritis initiated with collagen (type II), not on drugtherapy. Aspirin and PCO were suspended with 0.04% Tween-20 for oraldosing, after fasting animals overnight. Dose administered was 30 mg/kg.

Table 9 shows that PCO was virtually innocuous to the stomach even whengiven at 20 times an effective dose. This high dose (300 mg/kg) washowever the effective dose for aspirin in the standard anti-arthriticassay (adjuvant-induced rat polyarthritis), at which dose aspirin cancause considerable gastric damage.

TABLE 7 Mean Differences Days 8-15 †Rear †Rear Ft. Paw Gp Treatment N/gpL. Paw R. Paw* Inflam. ΔWt i None 4 1.77 mm 1.62 mm 2.5+ +10 gm ii OliveOil only 3 1.75 1.75 2.3+ +12 iii PCO/O.O 4 0.32 1.04 0.6+ +20 ivSeatone 4 0.74 1.11 0.8+ +12 (300 mg/kg) v Nurofen 3 0.82 1.48 0.7 + +15(40 mg/kg) % Inhibition Differences Days 15-18 (Rebound) Rear Rear FrontFront Gp L. paw R. paw paw Rear L. paw Rear R. paw paw ΔWt. i — — — 0.250.44 0.5+ −03 ii 01 (−8)  08 0.05 0.55 0 +02 iii 82 36 76 0.76 0.34 ++04 iv 58 31 68 0.64 0.22 1.2+ +03 v 54 07 72 0.38 0.38 1.1+ +05 *Rightpaw was pre-inflamed from original sensitising injection of collagen(II) in Freund's adjuvant. Note: Significant rebound in Gps (iii) and(iv) on ceasing dosing.

TABLE 8 Day 14: Signs arthritis Rear paw inflammation* †Left †RightFront Day Day rear rear paw ΔWt(0- Gp Treatment N/Gp 2 4 paw paw inflam.14) I None 4 1.41 mm 1.27 2.0 mm 1.75 mm 2.2+ +07 gm II Seatone p.o.x154 1.07 0.93 1.14 1.51 2+ +08 III PCO p.o.x15 4 1.06 0.80 1.25 1.69 0.8++13 IV Nurofen p.o.x15 3 1.48 1.52 1.35 1.72 2.5+ +12 V ATM s.c.x8 31.00 0.68 0.47 0.70 1.3+ (+24) VI Oil/Cineole t.d.x5 4 N.D. 1.84 1.301.9+ +02 VII PCO/Oil Cineole t.d.x5 4 N.D. 0.40 1.49 0.9+ +08 “Rebound”(Day 18) Percent Inhibitions Left Right Front †Left †Right Front Gp rearpaw rear paw paw Inflam. 2 4 rear paw rear paw paw inflam. I — — — — —II 24% 27% 43% 14% 09% III 25 37 37 03 64 IV (−05) (−20) 32 02 (−14) V29 46 76 60 41 VI N.D. 08 26 14 VII 0.57 0.29 1.4+ N.D. 80** 15** 59***Inflamed by injection of collagen (II) with a non-arthritogenicadjuvant to sensitise animals. **Note significant rebound.

TABLE 9 Gastric lesion index RATS (π = 3/gp) Aspirin PCO Low arthritis,Day 32 41 0 High arthritis, Day 15 82 0 High arthritis with 0.15 NHCl* >112 06 * HCl co-administered orally to stimulate acid secretionunder stress etc.

REFERENCES

1. Cullen, J. C., Flint, M. H. and Leider, J. (1975). N. Z. Med. J. 81:260-261.

2. Miller, T. E. and Ormrod, D. J. (1980). N. Z. Med. J. 92: 187-193.

3. Winter, C. A., Risely, E. A. and Nuss, G. W. (1962). Proc. Soc. Exp.Biol. Med. 111: 544-547.

4. Rainsford, K. D. and Whitehouse, M. W. (1980). Arzneim.-Forsch./DrugRes. 30 (ii), 2128-2132.

5. Gibson, R. G., Gibson, S. L. M., Conway, V. and Chappel, D. (1980).The Practitioner224:955-960.

6. Whitehouse, M. W. “Adjuvant-induced Polyarthritis in Rats”, inHandbook of Animal Models for the Rheumatic Diseases, Vol. 1, pages3-16, Editors R. A. Greenwald and H. S. Diamond, CRC Press, Inc., BocaRaton, Fla., USA.

What is claimed is:
 1. A method of anti-inflammatory treatment of ahuman or animal patient, which comprises administration to the patientof an effective amount of a lipid extract of Perna canaliculus orMytilus edulis rich in non-polar lipids, wherein said lipid extract isprepared by supercritical fluid extraction.
 2. A method according toclaim 1, wherein cryogenic fluid CO₂ is used in said supercritical fluidextraction.
 3. A method according to claim 1 or claim 2, wherein saidadministration is oral or subcutaneous administration.
 4. A methodaccording to claim 1 or claim 2, wherein said administration istransdermal administration.
 5. A method according to claim 1 or 2,wherein said anti-inflammatory treatment is treatment of arthritis,including rheumatoid arthritis and osteoarthritis.
 6. Ananti-inflammatory composition comprising a lipid extract of Pernacanaliculus or Mytilus edulis rich in non-polar lipids as an activecomponent thereof, wherein said lipid extract is prepared bysupercritical fluid extraction, together with one or morepharmaceutically acceptable carriers and/or diluents.
 7. A compositionaccording to claim 6, wherein cryogenic fluid CO₂ is used in saidsupercritical fluid extraction.
 8. A composition according to claim 6 orclaim 7, wherein said composition is formulated for oral or subcutaneousadministration of said active component.
 9. A composition according toclaim 6 or claim 7, wherein said composition is formulated fortransdermal administration of said active component.
 10. A compositionaccording to claim 9, comprising an ointment or lotion base or vehicle,and optionally a skin penetration enhancing agent.
 11. A compositionaccording to claim 10, wherein said base or vehicle comprises an oil.12. A composition according to claim 11, wherein said oil is olive oilor emu oil.
 13. A composition according to claim 10, which includes askin preparation enhancing agent.
 14. A composition according to claim13, wherein said agent is cineole or limonene.